An Improved Approach to Identify Irradiated Spices Using Electronic Nose, FTIR, and EPR Spectroscopy C: Food Chemistry
Bhaskar Sanyal, Jae-Jun Ahn, Jeong-Hwan Maeng, Hyun-Kyu Kyung, Ha-Kyeong Lim, Arun Sharma, and Joong-Ho Kwon
Changes in cumin and chili powder from India resulting from electron-beam irradiation were investigated using 3 analytical methods: electronic nose (E-nose), Fourier transform infrared (FTIR) spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. The spices had been exposed to 6 to 14 kGy doses recommended for microbial decontamination. E-nose measured a clear difference in flavor patterns of the irradiated spices in comparison with the nonirradiated samples. Principal component analysis further showed a dose-dependent variation. FTIR spectra of the samples showed strong absorption bands at 3425, 3007 to 2854, and 1746 cm−1 . However, both nonirradiated and irradiated spice samples had comparable patterns without any noteworthy changes in functional groups. EPR spectroscopy of the irradiated samples showed a radiation-specific triplet signal at g = 2.006 with a hyper-fine coupling constant of 3 mT confirming the results obtained with the E-nose technique. Thus, E-nose was found to be a potential tool to identify irradiated spices.
Abstract:
Keywords: detection, electronic nose, EPR spectroscopy, food irradiation; FTIR
Practical Application: Identification of irradiated food is of paramount importance to government regulatory bodies, the food irradiation industry, and consumers as there is increasing demand for irradiated food in international trade. In this study, detection of irradiated spices was investigated with an aim to develop a simple and rapid technique using electronic nose. Fourier transform infrared analysis was performed to know the changes in functional groups after irradiation. The accuracy and validity of electronic nose detection of prior irradiation was endorsed by electron paramagnetic resonance spectroscopy.
Introduction Spices from India have been recognized worldwide for their flavor, aroma, and medicinal properties. However, spices represent a potential source of microbial contamination for foodstuffs to which they are added. The exposure of spices to a high level of natural contamination by mesophylic, sporogenic, and asporogenic bacteria, hyphomycetes, and fecal coliforms during harvesting and storage is recognized as a realistic probability (Bendini and others 1998). Moreover, there is always a chance that the spices could be seriously contaminated by air- and soil-borne bacteria, fungi, and insects because most of the spices are dried in open air. Spices and herbs are currently treated with ionizing radiation to eliminate microbial contamination. It has been unambiguously confirmed that treatment with ionizing energy is more effective against bacteria than thermal treatment, and it does not leave chemical residues in the food product (Tjaberg and others 1972; Thayer and others 1996). Food irradiation technology is commercially employed in more than 55 countries around the world (IAEA 2013), and increasing amounts of irradiated food continue to circulate into the international trade market (Kume and Todoriki 2013; Parlatoa and MS 20140735 Submitted 5/1/2014, Accepted 7/7/2014. Authors Sanyal, Ahn, Maeng, Kyung, Lim, Sharma, and Kwon are with School of Food Science & Biotechnology, Kyungpook Natl. Univ., Daegu, 702–701, Korea. Authors Sanyal and Sharma are with Food Technology Div., Bhabha Atomic Research Center, Trombay, Mumbai, 400 085, India. Direct inquiry to author Joong-Ho Kwon (E-mail: [email protected]).
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others 2014). Use of accelerated electrons from linear accelerators is gradually increasing to irradiate food commodities because of their ability to process a large quantity of food within a short period of time. However, various national and international regulations with mandatory labeling requirements restrict the general use of this technology. The acceptability of irradiated food commodities needs reliable identification methods to enforce regulations and make possible traceability. A large number of research publications are available in the literature on the detection of irradiated foods treated with γ radiation (Sanyal and others 2009; Kwon and others 2013). However, very few reports deal with identifying irradiated food exposed to accelerated electrons. In principle, the effects of photon and/or electron beam irradiation on foods are similar in nature, but development of reliable and simple methods is of paramount interest to distinguish nonirradiated food samples from those exposed to the various allowed forms of ionizing energies. The interactions between biological materials and different forms of energy are very complex and depend on the irradiation and postirradiation conditions, which makes the detection of irradiated food a challenging task (Sanyal and others 2012). Various techniques, classified into 3 basic categories such as chemical, physical, and biological, have been studied to address this problem (Chung and others 2004). For some of these methods, international standards, such as the European standards by the European Committee for Standardization (CEN), have been formulated and adopted by the Codex Alimentarius Commission as Codex Standards. These include methods based on EPR spectroscopy R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12571 Further reproduction without permission is prohibited
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Figure 1–E-nose volatile patterns (fingerprints) of electron beam-irradiated spices.
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Detection of irradiated spices by E-nose, FT-IR, and EPR . . . these methods require sophisticated equipment and the procedures for the analysis are time consuming. Therefore, there is a need to find and develop simple tests that do not require expensive equipment for screening food items. The suspected samples can further be investigated by more reliable methods if necessary (Khan and Delinc´ee 1998).
Figure 3–FT-IR spectra of electron beam-irradiated spices.
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pertaining to foods containing bone tissue (EN1786 1997), cellulose (EN1787 2000), and crystalline sugar (EN13708 2001). Thermoluminescence of foods contaminated with silicate minerals (EN1788 2001), or gas chromatographic analysis of foods containing lipid-derived radiolytic products have also been recognized as useful detection methods. However, all
Detection of irradiated spices by E-nose, FT-IR, and EPR . . .
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of the sample tube was then sealed with plastic film. EPR measurements were performed according to the European standard, EN1787 (2000), using a X-band EPR spectrometer (JES-FE200; Jeol Co., Tokyo, Japan) at room temperature. The EPR signal height was computed using ESPRIT-425 software (Jeol Co.) as the peak-to-peak amplitude of the first derivative spectrum, whereas the signal intensity was presented in arbitrary units per unit sample weight (AU/mg). The EPR measurements were under the following experimental conditions: microwave power, 0.4 mW; microwave frequency, 9.19 to 9.21 GHz; center field, 337 ± 1 mT; sweep width, 10 to 25 mT; modulation frequency, 100 kHz; Materials and Methods modulation width, 1 to 2 mT; amplitude, 50 to 400; sweep time, Samples 30 s; and time constant, 0.03 s. The g-values were calculated using Cumin powder (2 packets) from 2 different companies (Everest an internal Mn (II) standard attached to the ESR cavity. and Ramdev in India) and red chili powder (1 packet) of Everest make were purchased from a local market in Mumbai, India. These Statistical analysis spices were handpicked from carefully selected crops and sunAll experiments were conducted at least in triplicate and the dried before being ground to the perfect texture by accurately data analysis was performed using Origin 8.6 (Microcal Software calibrated machines during the process of manufacturing. It was Inc., Northampton, Mass., U.S.A.) and the SAS program (version also ensured that the samples had not been subjected to ionizing 9.3; SAS Institute, Cary, N.C., U.S.A.). radiation before marketing. All the spice powders without any prior treatment were packed in polyvinylchloride (PVC) film and stored at room temperature. The samples were identified by this Results and Discussion code: cumin (Everest): EC, cumin (Ramdev): RC, and red chili E-nose analysis of irradiated spices (Everest): TR. The electronic nose is an instrument that comprises an array In this study, a new technique based on electronic nose (Enose) was employed to study the changes in the flavor patterns of 2 Indian spice samples (cumin and chili powder) exposed to electron beam at commercially relevant doses. Fourier transform infrared (FTIR) analysis was carried out to study the changes in functional groups induced by ionizing radiation. Finally, the irradiated samples were investigated using EPR spectroscopy to identify radiation-induced paramagnetic centers to confirm the reliability of the E-nose technique in detecting irradiated samples.
Irradiation All the 3 samples were distributed into 4 parts using polyethylene pouches. One part of each sample was kept nonirradiated as the control and the remaining 3 parts were irradiated at 6, 10, and 14 kGy doses using electron beam from an electron accelerator (UELV-10-10s, 10 MeV; EB-Tech, Daejeon, South Korea) at room temperature. Absorbed doses were measured using an alanine-electron paramagnetic resonance dosimetry system, with an EMS 104 EPR analyzer (Bruker Biospin, Rheinstetten, Germany). Evaluation of volatile compounds by electronic nose A zNose (Electronic Sensor Technology, Newbury Park, Calif., U.S.A.), equipped with a surface acoustic wave (SAW) sensor and VaporPrintTM (Misrosense 4.88) software, was used to evaluate changes in volatile compounds. A 1-g sample was added to a 40-mL vial (Supelco, Bellefonte, Pa., U.S.A.) fitted with a Teflon septum (PTFE/silicone, Supelco), sealed, and placed at room temperature for overnight to obtain the headspace equilibrium. The equipment was operated at a SAW sensor temperature of 30 °C; column, 60 °C; valve, 120 °C; inlet, 150 °C; and trap, 220 °C. Principal component analysis (PCA) was used to identify the trend in the numerical data (Ahn and others 2013b). FT-IR analysis The spice sample was ground with a spectroscopic-grade potassium bromide (KBr) powder and then pressed into 1-mm pellets. The analysis was conducted on a Perkin-Elmer spectrophotometer (model Spectrum GX; Norwalk, Conn., U.S.A.) equipped with Perkin-Elmer AutoImage microscope in a frequency range of 400 to 4000 cm−1 . Electron paramagnetic resonance spectroscopy The spice samples were directly used for the EPR analysis without prior sample preparation. Approximately 0.1 g of sample was placed in a quartz EPR tube (5-mm diameter). The open end C1660 Journal of Food Science r Vol. 79, Nr. 9, 2014
of electronic chemical sensors of partial specificity and an appropriate pattern-recognition system, capable of recognizing simple or complex odors (Gardner and Bartlett 1993). The instrument registers the electrical signals generated due to the alteration of the state of the sensors after the chemical interaction with the odor compounds. The characterization of gaseous samples as a global fingerprint or chemical image can be conducted using the combined responses coupled with a suitable pattern-recognition system (Ruiz-Altisent and others 2010). E-nose analysis has been established as a nondestructive technique to characterize various quality parameters of foods depending on their odor patterns. To detect and evaluate the changes in flavor patterns of various irradiated foods, the metal oxide sensor (MOS), which responds to specific substances, and conducting polymer sensor were mainly used (Kim and Noh 1999). However, according to Han and others (2009) the electronic nose equipped with the SAW sensor was capable to determine the flavor patterns of γ -irradiated raw oyster within a short period. The Z-nose based on surface acoustic wave (SAW) sensor has the advantage of a rapid analysis and simple preprocessing of sample, although the measured data are greatly dependent upon the retention time (Cho and Noh 2002). In this study, the effect of flavor pattern of the irradiated spices was analyzed with a SAW sensor. The Vapor Print (polar graphs of peaks) to frequency pattern was obtained by differentiation of frequency chromatogram and transformation into a cycle based on total retention time as shown in Figure 1. Different electronic fingerprints were observed in the irradiated spice samples compared to the nonirradiated ones. To obtain a clearer trend in the numerical data, the PCA technique was used. Figure 2 shows the E-nose analysis results from 2 PCs. The control and irradiated samples formed distinct groups (circled) depending on the applied doses. A prominent difference in the volatile compound profiles of the samples was found out upon irradiation. There were some overlaps in the groups of the PCA score plots for the irradiated samples. However, a clear distinction between nonirradiated and irradiated samples was observed for all the samples. The most promising results were observed for red chili powder (TR). This distinctive fingerprint
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Detection of irradiated spices by E-nose, FT-IR, and EPR . . . corresponding to C–O, C–H stretching (Cael and others 1975). Both nonirradiated and irradiated spice samples had a comparable pattern without any noteworthy changes in FTIR spectra. However, in the case of food samples rich in glycosidic bonds a strong absorption band at 2337 cm−1 was detected after γ irradiation and this band has been shown to result from entrapment of free CO2 gases within the cross-linked polymers when exposed to ionizing radiation (Kumar and D’Souza 2008). On the other hand, Byun and others (2008) reported no change in the FTIR spectrum of control and irradiated β-glucan. Relleve and others (2005) confirmed with FTIR spectra that there are no functional group changes even at 100 kGy in k-carrageenan. Like γ -irradiated food samples our results also showed that all the absorption values of electron beam-irradiated spice samples were similar to those of the nonirradiated ones confirming no major change in the functional groups. Therefore, in view of detecting irradiated samples, FTIR was found to be not suitable, leading to the requirement of studies using an established detection technique to confirm the results obtained from the E-nose method.
FTIR spectroscopy of irradiated spices Carbohydrates are one of the major constituents in spices. Deformation of carbohydrate by irradiation might occur because of the splintering of glycosidic bonds (von Sonntag 1980). To compare the changes in chemical structure of nonirradiated and electron beam-irradiated spice samples, FTIR spectra were analyzed. Figure 3 shows FTIR spectra of the spice samples before and after irradiation, recorded in the absorption bands at 400 to 4000 cm−1 . Strong absorption bands were observed for all the samples at 3425, 3007 to 2854, and 1746 cm−1 in the infrared region of the spectra corresponding to functional groups such as the stretching-absorption bands of poly-OH, asymmetric sketching vibration of methyl groups (C–H), and C=O sketching vibration (carbonyl or amide), respectively (Mathlouthi and Koenig 1986; Guitierrez and others 1996; Byun and others 2008). The main absorptions were approximately detected at 2925 and 2854 cm−1 , and there were also absorptions at 1457 and 722 cm−1 , which indicated the probability of a long linear aliphatic chain (John 2000). In addition, a weak absorption band at 1160 cm−1 was observed
EPR spectroscopy of irradiated spices E-nose analysis showed a clear distinction in flavor patterns between irradiated and non-irradiated spice samples but was not sufficient to give a clear verdict on radiation treatment. FTIR results showed no radiation-induced functional group transformations. Therefore, to confirm the irradiation status of the spices, an investigation on radiation-induced paramagnetic centers were conducted using EPR spectroscopy. Figure 4 shows the EPR spectra of all 3 spice samples before and after the radiation treatment. The nonirradiated spectra were characterized by a central line at g = 2.006, with a line width (Bpp ) of 0.54 mT as a native signal, possibly due to the photooxodation of the existing polyphenols. Several reports have suggested that these free radicals are semiquinone radicals produced by the oxidation of plant polyphenolics (Scewartz and others 1972) or lignin (Maloney and others 1992; Tabner and Tabner 1994). In the case of both the cumin samples, a sextet signal was observed showing a trace of Mn2+ as depicted in Figure 4. However, the EPR lines of Mn2+ have been observed in many other foods and identified as
Figure 5–Normalized EPR intensity of the electron beam-irradiated spices with increasing radiation doses.
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of volatile profile can represent the differences and similarities of odor patterns from different food samples (Kim and others 2004). In the field of identification of irradiated foods, limited application of E-nose to screen γ -irradiated food samples have been found in the literature, such as detection of irradiated tomatoes (Winquist and others 1995), anchovy sauce (Kim and others 2004), red pepper powder (Lee and others 2004). A discrimination of the odor patterns of Korean radish kimchi containing irradiated red pepper powder has been reported by Lee and others (2005), showing the screening potential of E-nose analysis in secondary products with an irradiated ingredient. Similarly, the electron-beam–irradiated spice samples also showed an identical response in flavor patterns as compared to the γ -irradiated food samples with a marked difference from their nonirradiated counterparts. However, E-nose analysis for identification of irradiated food has been considered as a screening method because the radiation-induced changes in odor patterns were not radiation-specific detection markers. Therefore, employment of other analytical approaches was necessary to get a clear verdict on the irradiation status of the spice samples.
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Conclusion A distinct difference in the flavor patterns of irradiated spice samples was observed when comparing with nonirradiated ones using E-nose. PCA results further depicted dose-dependent groupings of the numerical data, confirming the ability of this technique to detect irradiated spices. All the irradiated samples were identified and the most prominent result was observed with the red chili sample. Both nonirradiated and irradiated spice samples showed a comparable pattern in FTIR spectra revealing no noteworthy changes in functional groups after irradiation. However, EPR spectra of the irradiated samples exhibited a clear distinction from the nonirradiated one with a radiation-induced paramagnetic center. Although the working principles of both the detection techniques, namely E-nose and EPR spectroscopy, were redundant in nature, their findings were similar and both the measurements showed promising results in identifying irradiated red chili. All the methods employed in this study showed similar changes in electron beam-irradiated spices similar to those observed in γ -irradiated samples reported in the literature. Here we report, probably for the first time, that E-nose analysis could be used as a novel approach to screen electron beam-irradiated spice samples; and the authenticity of the results could be confirmed by EPR spectroscopy.
Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2013R1A1A4A03006993).
References Ahn JJ, Akram K, Kim HK, Kwon JH. 2013a. Electron spin resonance spectroscopy for the identification of irradiated food with complex ESR signals. Food Analytical Methods 6:301– 8. Ahn JJ, Akram K, Kwak JY, Jeong MS, Kwon JH. 2013b. Reliable screening of various food stuffs with respect to the irradiation status: a comparative study of different analytical techniques. Radiat Phys Chem 91:186–92. Bendini A, Galina Toschi T, Lercker G. 1998. Influence of γ irradiation and microwaves on the linear unsaturated hydrocarbon fraction in spices. Z Lebensm Unters Forsch 207: 214–8. Byun E, Kim J, Sung N, Choi J, Lim S, Kim K. 2008. Effects of γ irradiation on the physical and structural properties of β-glucan. Radiat Phys Chem 77:781–6. Cael JJ, Koenig JL, Blackwell J. 1975. Infrared and Raman spectroscopy of carbohydrates. Part VI: Normal coordinate analysis of V-amylose. Biopolymers 14:1885–903. Cho YS, Noh BS. 2002. Quality evaluation of dried laver (Porphyra yezoensis Ueda) using electronic nose based on metal oxide sensor or GC with SAW sensor during storage. Korean J Food Sci Technol 34:947–53. Chung HW, Delinc´ee H, Han SB, Hong JH, Kim HY, Kim MC, Byun MW, Kwon JH. 2004. Trials to identify irradiated chestnut (Castanea bungena) with different analytical techniques. Radiat Phys Chem 71:181–4. EN1786. 1997. Foodstuffs – Detection of irradiated food containing bone: analysis by electron paramagnetic resonance; European Committee of Standardization (CEN), Brussels, Belgium. EN1787. 2000. Foodstuffs – Detection of irradiated food containing cellulose: analysis by EPR; European Committee of Standardization (CEN), Brussels, Belgium. EN13708. 2001. Foodstuffs – Detection of irradiated food containing crystalline sugar: analysis by EPR; European Committee of Standardization (CEN), Brussels, Belgium. EN1788. 2001. Foodstuffs – Thermoluminescence detection of irradiated food from which silicate minerals can be isolated; European Committee of Standardization (CEN), Brussels, Belgium. Gardner JW, Bartlett PN. 1993. Brief history of electronic nose. Sensor Actuat B-Chem 18:211– 20. Gutierrez A, Prieto A, Martlnez AT. 1996. Structural characterization of extracellular polysaccharides produced by fungi from the genus Pleurotus. Carbohydr Res 281: 143–54. Han IJ, Park JS, Choi JI, Kim JH, Song, BS, Yoon Y, Byun MW, Chun SS, Lee JW. 2009. Change in flavor patterns of γ -irradiated raw oyster and oyster cooking drip determined using and electronic nose. Korean J Aquat Sci 42:209–14. IAEA. 2013. Food authorization database. Available from: http://nucleus.iaea.org/. Accessed 2014 April 14. John C. 2000. Interpretation of infrared spectra: a practical approach. In: Meyers RA, editor. Encyclopedia of analytical chemistry. New York, NY: Wiley & Sons, Ltd: p 10815–37. Khan HM, Delinc´ee H. 1998. Detection of irradiation treatment of foods using DNA ‘Comet Assay’. Radiat Phys Chem 52:141–4. Kim JH, Noh BS. 1999. Detection of irradiation treatment for red peppers by an electronic nose using conducting polymer sensors. Food Sci Biotechnol 8:207–9. Kim JH, Ahn HJ, Yook HS, Kim KS, Rhee MS, Ryu GH, Byun MW. 2004. Color, flavor, and sensory characteristics of γ -irradiated salted and fermented anchovy sauce. Radiat Phys Chem 69:179–87. Kumar J, D’Souza SF. 2008. Preparation of PVA membrane for immobilization of GOD for glucose biosensor. Talanta 75:183–8. Kume T, Todoriki S. 2013. Food irradiation in Asia, the European Union, and the United States: a status update. Radioisotopes 62(5):291–9. Kwon JH, Ahn JJ, Akram K, Son IJ, Lee SO. 2013. Characterization of radiation-induced luminescence properties and free radicals for the identification of different γ -irradiated teas. Anal Bioanal Chem, 405:4225–34. Lee J, Kausar T, Kim BK, Kwon JH. 2008. Detection of γ -irradiated sesame seeds before and after roasting by analyzing photostimulated luminescence, thermoluminescence and electron spin resonance. J Agric Food Chem 56:7184–8. Lee JH, Sung TW, Lee KT, Kim MR. 2004. Effect of γ -irradiation on color, pungency, and volatiles of Korean red pepper powder. J Food Sci 69:C585–92. Lee JH, Lee KT, Kim MR. 2005. Effect of γ -irradiated red pepper powder on the chemical and volatile characteristics of kakdugi, a Korean traditional fermented radish kimchi. J Food Sci 70:C441–7. Maloney DR, Tabner BJ, Tabner VA. 1992. An electron spin resonance study of γ irradiated fruits. Radiat Phys Chem 3:309–14. Mathlouthi M, Koenig JL. 1986. Vibrational spectra of carbohydrates. Adv Carbohydr Chem Biochem 44:7–89. Parlatoa A, Giacomarrab M, Galatic A, Crescimanno M. 2014. ISO 14470:2011 and EU legislative background on food irradiation technology: The Italian attitude. Trends Food Sci Technol 38:60–74. Raffi J, Stevenson MH, Kent M, Thiery JM, Belliardo JJ. 1992. Intercomparison on electron spin resonance identification of irradiated foodstuffs. Int J Food Sci Technol 27: 111–24. Relleve L, Nagasawa N, Luan LQ, Yagi T, Aranilla C, Abad L. 2005. Degradation of carrageenan by radiation. Polym Degrad Stabil 87:403–10. Ruiz-Altisent M, Ruiz-Garcia L, Moreda GP, Lu R, Hernandez-Sanchez N, Correa EC, Diezma B, Nicolai B, Garcia-Ramos J. 2010. Sensors for product characterization and quality of specialty crops—a review. Comput Electron Agric 74:176–94. Sanyal B, Sharma A. 2009. A new electron paramagnetic resonance method to identify irradiated soybean. J Food Sci 74:N57–64. Sanyal B, Chawla SP, Sharma A. 2009. An improved method to identify irradiated rice by EPR spectroscopy and thermoluminescence measurements. Food Chem 116:526–34.
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independent of radiation processing (Sanyal and Sharma 2009). In the case of all the spice samples, complex EPR spectra were observed, with an increase in the signal intensity of the existing weak singlet (g = 2.006) after the exposure to electron beam at 6, 10, and 14 kGy doses. Raffi and others (1992) and Ahn and others (2013a) previously had reported similar observations, where an intense signal was noticed in the spectrum of irradiated spices; the relatively high intensity was attributed to the irradiation treatment. The line width (Bpp ) of the EPR signal was observed to increase from 0.54 to 0.93 mT possibly because of the induction of multiple paramagnetic centers in the matrix of the samples. The exposure to electron beam leads to another paramagnetic species (triplet signal) at the same g value as that of nonirradiated samples, with a hyperfine coupling constant (hfcc) of 3 mT. This signal was attributed to cellulose radical induced by the ionizing radiation as a consequence of the cleavage of the cellulose polymer chain. The triplet was found to be more prominent in the case of irradiated red chili compared to the cumin samples. Yordanov and Gancheva (2000) and Lee and others (2008) have reported similar observations for γ -irradiated white pepper and sesame seeds, respectively. The signature of the cellulose radical was also found in γ -irradiated soybeans as reported in our earlier communication (Sanyal and Sharma 2009). The triplet signal of the cellulosic radical is a well-established marker for radiation-processed foods according to EN1787 (2000). The effect of increasing the radiation dose on the EPR spectra of the spice samples was studied. At a dose of 14 kGy, the triplet of cellulose was most prominent with maximum signal intensity. The peak to peak intensity of the EPR central line was observed to be significantly related to the radiation dose as shown in Figure 5. The dotted lines represent a linear fit with R2 values of 0.9892, 0.9441, and 0.9702 for EC, RC, and TR samples, respectively. The EPR spectral features of the electron beam-irradiated spice samples were identical to those of the γ -irradiated food samples reported in the literature (Lee and others 2008; Sanyal and Sharma 2009). However, our irradiated spice samples showed distinct changes in EPR spectra with respect to the nonirradiated samples, thus confirming the results obtained with E-nose analysis.
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Sanyal B, Chatterjee S, Variyar PS, Sharma A. 2012. Application of EPR spectroscopy to identify irradiated Indian medicinal plant products. J Food Sci 77:C710–8. Scewartz HM, Bolton JR, Brog DC. 1972. Biological applications of electron spin resonance. New York, NY: Wiley & Sons, Ltd. Tabner BJ, Tabner VA. 1994. Electron spin resonance spectra of γ -irradiated citrus fruits, skin, skin components and stalks. Int J Food Sci Technol 29:143–52. Thayer DW, Josephson ES, Brynjolfsson A, Giddings GG. 1996. Radiation pasteurization of food. Council Agric Sci Technol 7:1–10. Tjaberg TB, Underdal B, Lunde G. 1972. The effect of ionizing radiation on the microbial content and volatile constituents of spices. J Appl Bacteriol 35:473–8.
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von Sonntag C. 1980. Free-radicals reactions of carbohydrates as studied by radiation techniques. Adv Carbohydr Chem Biol 37:7–77. Winquist F, Arwin H, Lund E, Forster R, Day C, Lundstrom I. 1995. Screening of irradiated tomatoes by means of an electronic nose. In Proceedings of Transducers’ 95, Euro sensors IX, the 8th International Conference on Solid- state Sensors and Actuators and Euro sensors IX; June 25–29, pp 691–669; Stockholm, Sweden. Yordanov ND, Gancheva V. 2000. A new approach for extension of the identification period of irradiated cellulose-containing food stuffs by EPR spectroscopy. Appl Radiat Isotopes 52:195–8.
Bhaskar Sanyal, Jae-Jun Ahn, Jeong-Hwan Maeng, Hyun-Kyu Kyung, Ha-Kyeong Lim, Arun Sharma, and Joong-Ho Kwon
Changes in cumin and chili powder from India resulting from electron-beam irradiation were investigated using 3 analytical methods: electronic nose (E-nose), Fourier transform infrared (FTIR) spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. The spices had been exposed to 6 to 14 kGy doses recommended for microbial decontamination. E-nose measured a clear difference in flavor patterns of the irradiated spices in comparison with the nonirradiated samples. Principal component analysis further showed a dose-dependent variation. FTIR spectra of the samples showed strong absorption bands at 3425, 3007 to 2854, and 1746 cm−1 . However, both nonirradiated and irradiated spice samples had comparable patterns without any noteworthy changes in functional groups. EPR spectroscopy of the irradiated samples showed a radiation-specific triplet signal at g = 2.006 with a hyper-fine coupling constant of 3 mT confirming the results obtained with the E-nose technique. Thus, E-nose was found to be a potential tool to identify irradiated spices.
Abstract:
Keywords: detection, electronic nose, EPR spectroscopy, food irradiation; FTIR
Practical Application: Identification of irradiated food is of paramount importance to government regulatory bodies, the food irradiation industry, and consumers as there is increasing demand for irradiated food in international trade. In this study, detection of irradiated spices was investigated with an aim to develop a simple and rapid technique using electronic nose. Fourier transform infrared analysis was performed to know the changes in functional groups after irradiation. The accuracy and validity of electronic nose detection of prior irradiation was endorsed by electron paramagnetic resonance spectroscopy.
Introduction Spices from India have been recognized worldwide for their flavor, aroma, and medicinal properties. However, spices represent a potential source of microbial contamination for foodstuffs to which they are added. The exposure of spices to a high level of natural contamination by mesophylic, sporogenic, and asporogenic bacteria, hyphomycetes, and fecal coliforms during harvesting and storage is recognized as a realistic probability (Bendini and others 1998). Moreover, there is always a chance that the spices could be seriously contaminated by air- and soil-borne bacteria, fungi, and insects because most of the spices are dried in open air. Spices and herbs are currently treated with ionizing radiation to eliminate microbial contamination. It has been unambiguously confirmed that treatment with ionizing energy is more effective against bacteria than thermal treatment, and it does not leave chemical residues in the food product (Tjaberg and others 1972; Thayer and others 1996). Food irradiation technology is commercially employed in more than 55 countries around the world (IAEA 2013), and increasing amounts of irradiated food continue to circulate into the international trade market (Kume and Todoriki 2013; Parlatoa and MS 20140735 Submitted 5/1/2014, Accepted 7/7/2014. Authors Sanyal, Ahn, Maeng, Kyung, Lim, Sharma, and Kwon are with School of Food Science & Biotechnology, Kyungpook Natl. Univ., Daegu, 702–701, Korea. Authors Sanyal and Sharma are with Food Technology Div., Bhabha Atomic Research Center, Trombay, Mumbai, 400 085, India. Direct inquiry to author Joong-Ho Kwon (E-mail: [email protected]).
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others 2014). Use of accelerated electrons from linear accelerators is gradually increasing to irradiate food commodities because of their ability to process a large quantity of food within a short period of time. However, various national and international regulations with mandatory labeling requirements restrict the general use of this technology. The acceptability of irradiated food commodities needs reliable identification methods to enforce regulations and make possible traceability. A large number of research publications are available in the literature on the detection of irradiated foods treated with γ radiation (Sanyal and others 2009; Kwon and others 2013). However, very few reports deal with identifying irradiated food exposed to accelerated electrons. In principle, the effects of photon and/or electron beam irradiation on foods are similar in nature, but development of reliable and simple methods is of paramount interest to distinguish nonirradiated food samples from those exposed to the various allowed forms of ionizing energies. The interactions between biological materials and different forms of energy are very complex and depend on the irradiation and postirradiation conditions, which makes the detection of irradiated food a challenging task (Sanyal and others 2012). Various techniques, classified into 3 basic categories such as chemical, physical, and biological, have been studied to address this problem (Chung and others 2004). For some of these methods, international standards, such as the European standards by the European Committee for Standardization (CEN), have been formulated and adopted by the Codex Alimentarius Commission as Codex Standards. These include methods based on EPR spectroscopy R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12571 Further reproduction without permission is prohibited
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Figure 3–FT-IR spectra of electron beam-irradiated spices.
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pertaining to foods containing bone tissue (EN1786 1997), cellulose (EN1787 2000), and crystalline sugar (EN13708 2001). Thermoluminescence of foods contaminated with silicate minerals (EN1788 2001), or gas chromatographic analysis of foods containing lipid-derived radiolytic products have also been recognized as useful detection methods. However, all
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of the sample tube was then sealed with plastic film. EPR measurements were performed according to the European standard, EN1787 (2000), using a X-band EPR spectrometer (JES-FE200; Jeol Co., Tokyo, Japan) at room temperature. The EPR signal height was computed using ESPRIT-425 software (Jeol Co.) as the peak-to-peak amplitude of the first derivative spectrum, whereas the signal intensity was presented in arbitrary units per unit sample weight (AU/mg). The EPR measurements were under the following experimental conditions: microwave power, 0.4 mW; microwave frequency, 9.19 to 9.21 GHz; center field, 337 ± 1 mT; sweep width, 10 to 25 mT; modulation frequency, 100 kHz; Materials and Methods modulation width, 1 to 2 mT; amplitude, 50 to 400; sweep time, Samples 30 s; and time constant, 0.03 s. The g-values were calculated using Cumin powder (2 packets) from 2 different companies (Everest an internal Mn (II) standard attached to the ESR cavity. and Ramdev in India) and red chili powder (1 packet) of Everest make were purchased from a local market in Mumbai, India. These Statistical analysis spices were handpicked from carefully selected crops and sunAll experiments were conducted at least in triplicate and the dried before being ground to the perfect texture by accurately data analysis was performed using Origin 8.6 (Microcal Software calibrated machines during the process of manufacturing. It was Inc., Northampton, Mass., U.S.A.) and the SAS program (version also ensured that the samples had not been subjected to ionizing 9.3; SAS Institute, Cary, N.C., U.S.A.). radiation before marketing. All the spice powders without any prior treatment were packed in polyvinylchloride (PVC) film and stored at room temperature. The samples were identified by this Results and Discussion code: cumin (Everest): EC, cumin (Ramdev): RC, and red chili E-nose analysis of irradiated spices (Everest): TR. The electronic nose is an instrument that comprises an array In this study, a new technique based on electronic nose (Enose) was employed to study the changes in the flavor patterns of 2 Indian spice samples (cumin and chili powder) exposed to electron beam at commercially relevant doses. Fourier transform infrared (FTIR) analysis was carried out to study the changes in functional groups induced by ionizing radiation. Finally, the irradiated samples were investigated using EPR spectroscopy to identify radiation-induced paramagnetic centers to confirm the reliability of the E-nose technique in detecting irradiated samples.
Irradiation All the 3 samples were distributed into 4 parts using polyethylene pouches. One part of each sample was kept nonirradiated as the control and the remaining 3 parts were irradiated at 6, 10, and 14 kGy doses using electron beam from an electron accelerator (UELV-10-10s, 10 MeV; EB-Tech, Daejeon, South Korea) at room temperature. Absorbed doses were measured using an alanine-electron paramagnetic resonance dosimetry system, with an EMS 104 EPR analyzer (Bruker Biospin, Rheinstetten, Germany). Evaluation of volatile compounds by electronic nose A zNose (Electronic Sensor Technology, Newbury Park, Calif., U.S.A.), equipped with a surface acoustic wave (SAW) sensor and VaporPrintTM (Misrosense 4.88) software, was used to evaluate changes in volatile compounds. A 1-g sample was added to a 40-mL vial (Supelco, Bellefonte, Pa., U.S.A.) fitted with a Teflon septum (PTFE/silicone, Supelco), sealed, and placed at room temperature for overnight to obtain the headspace equilibrium. The equipment was operated at a SAW sensor temperature of 30 °C; column, 60 °C; valve, 120 °C; inlet, 150 °C; and trap, 220 °C. Principal component analysis (PCA) was used to identify the trend in the numerical data (Ahn and others 2013b). FT-IR analysis The spice sample was ground with a spectroscopic-grade potassium bromide (KBr) powder and then pressed into 1-mm pellets. The analysis was conducted on a Perkin-Elmer spectrophotometer (model Spectrum GX; Norwalk, Conn., U.S.A.) equipped with Perkin-Elmer AutoImage microscope in a frequency range of 400 to 4000 cm−1 . Electron paramagnetic resonance spectroscopy The spice samples were directly used for the EPR analysis without prior sample preparation. Approximately 0.1 g of sample was placed in a quartz EPR tube (5-mm diameter). The open end C1660 Journal of Food Science r Vol. 79, Nr. 9, 2014
of electronic chemical sensors of partial specificity and an appropriate pattern-recognition system, capable of recognizing simple or complex odors (Gardner and Bartlett 1993). The instrument registers the electrical signals generated due to the alteration of the state of the sensors after the chemical interaction with the odor compounds. The characterization of gaseous samples as a global fingerprint or chemical image can be conducted using the combined responses coupled with a suitable pattern-recognition system (Ruiz-Altisent and others 2010). E-nose analysis has been established as a nondestructive technique to characterize various quality parameters of foods depending on their odor patterns. To detect and evaluate the changes in flavor patterns of various irradiated foods, the metal oxide sensor (MOS), which responds to specific substances, and conducting polymer sensor were mainly used (Kim and Noh 1999). However, according to Han and others (2009) the electronic nose equipped with the SAW sensor was capable to determine the flavor patterns of γ -irradiated raw oyster within a short period. The Z-nose based on surface acoustic wave (SAW) sensor has the advantage of a rapid analysis and simple preprocessing of sample, although the measured data are greatly dependent upon the retention time (Cho and Noh 2002). In this study, the effect of flavor pattern of the irradiated spices was analyzed with a SAW sensor. The Vapor Print (polar graphs of peaks) to frequency pattern was obtained by differentiation of frequency chromatogram and transformation into a cycle based on total retention time as shown in Figure 1. Different electronic fingerprints were observed in the irradiated spice samples compared to the nonirradiated ones. To obtain a clearer trend in the numerical data, the PCA technique was used. Figure 2 shows the E-nose analysis results from 2 PCs. The control and irradiated samples formed distinct groups (circled) depending on the applied doses. A prominent difference in the volatile compound profiles of the samples was found out upon irradiation. There were some overlaps in the groups of the PCA score plots for the irradiated samples. However, a clear distinction between nonirradiated and irradiated samples was observed for all the samples. The most promising results were observed for red chili powder (TR). This distinctive fingerprint
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Detection of irradiated spices by E-nose, FT-IR, and EPR . . . corresponding to C–O, C–H stretching (Cael and others 1975). Both nonirradiated and irradiated spice samples had a comparable pattern without any noteworthy changes in FTIR spectra. However, in the case of food samples rich in glycosidic bonds a strong absorption band at 2337 cm−1 was detected after γ irradiation and this band has been shown to result from entrapment of free CO2 gases within the cross-linked polymers when exposed to ionizing radiation (Kumar and D’Souza 2008). On the other hand, Byun and others (2008) reported no change in the FTIR spectrum of control and irradiated β-glucan. Relleve and others (2005) confirmed with FTIR spectra that there are no functional group changes even at 100 kGy in k-carrageenan. Like γ -irradiated food samples our results also showed that all the absorption values of electron beam-irradiated spice samples were similar to those of the nonirradiated ones confirming no major change in the functional groups. Therefore, in view of detecting irradiated samples, FTIR was found to be not suitable, leading to the requirement of studies using an established detection technique to confirm the results obtained from the E-nose method.
FTIR spectroscopy of irradiated spices Carbohydrates are one of the major constituents in spices. Deformation of carbohydrate by irradiation might occur because of the splintering of glycosidic bonds (von Sonntag 1980). To compare the changes in chemical structure of nonirradiated and electron beam-irradiated spice samples, FTIR spectra were analyzed. Figure 3 shows FTIR spectra of the spice samples before and after irradiation, recorded in the absorption bands at 400 to 4000 cm−1 . Strong absorption bands were observed for all the samples at 3425, 3007 to 2854, and 1746 cm−1 in the infrared region of the spectra corresponding to functional groups such as the stretching-absorption bands of poly-OH, asymmetric sketching vibration of methyl groups (C–H), and C=O sketching vibration (carbonyl or amide), respectively (Mathlouthi and Koenig 1986; Guitierrez and others 1996; Byun and others 2008). The main absorptions were approximately detected at 2925 and 2854 cm−1 , and there were also absorptions at 1457 and 722 cm−1 , which indicated the probability of a long linear aliphatic chain (John 2000). In addition, a weak absorption band at 1160 cm−1 was observed
EPR spectroscopy of irradiated spices E-nose analysis showed a clear distinction in flavor patterns between irradiated and non-irradiated spice samples but was not sufficient to give a clear verdict on radiation treatment. FTIR results showed no radiation-induced functional group transformations. Therefore, to confirm the irradiation status of the spices, an investigation on radiation-induced paramagnetic centers were conducted using EPR spectroscopy. Figure 4 shows the EPR spectra of all 3 spice samples before and after the radiation treatment. The nonirradiated spectra were characterized by a central line at g = 2.006, with a line width (Bpp ) of 0.54 mT as a native signal, possibly due to the photooxodation of the existing polyphenols. Several reports have suggested that these free radicals are semiquinone radicals produced by the oxidation of plant polyphenolics (Scewartz and others 1972) or lignin (Maloney and others 1992; Tabner and Tabner 1994). In the case of both the cumin samples, a sextet signal was observed showing a trace of Mn2+ as depicted in Figure 4. However, the EPR lines of Mn2+ have been observed in many other foods and identified as
Figure 5–Normalized EPR intensity of the electron beam-irradiated spices with increasing radiation doses.
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of volatile profile can represent the differences and similarities of odor patterns from different food samples (Kim and others 2004). In the field of identification of irradiated foods, limited application of E-nose to screen γ -irradiated food samples have been found in the literature, such as detection of irradiated tomatoes (Winquist and others 1995), anchovy sauce (Kim and others 2004), red pepper powder (Lee and others 2004). A discrimination of the odor patterns of Korean radish kimchi containing irradiated red pepper powder has been reported by Lee and others (2005), showing the screening potential of E-nose analysis in secondary products with an irradiated ingredient. Similarly, the electron-beam–irradiated spice samples also showed an identical response in flavor patterns as compared to the γ -irradiated food samples with a marked difference from their nonirradiated counterparts. However, E-nose analysis for identification of irradiated food has been considered as a screening method because the radiation-induced changes in odor patterns were not radiation-specific detection markers. Therefore, employment of other analytical approaches was necessary to get a clear verdict on the irradiation status of the spice samples.
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Detection of irradiated spices by E-nose, FT-IR, and EPR . . .
Conclusion A distinct difference in the flavor patterns of irradiated spice samples was observed when comparing with nonirradiated ones using E-nose. PCA results further depicted dose-dependent groupings of the numerical data, confirming the ability of this technique to detect irradiated spices. All the irradiated samples were identified and the most prominent result was observed with the red chili sample. Both nonirradiated and irradiated spice samples showed a comparable pattern in FTIR spectra revealing no noteworthy changes in functional groups after irradiation. However, EPR spectra of the irradiated samples exhibited a clear distinction from the nonirradiated one with a radiation-induced paramagnetic center. Although the working principles of both the detection techniques, namely E-nose and EPR spectroscopy, were redundant in nature, their findings were similar and both the measurements showed promising results in identifying irradiated red chili. All the methods employed in this study showed similar changes in electron beam-irradiated spices similar to those observed in γ -irradiated samples reported in the literature. Here we report, probably for the first time, that E-nose analysis could be used as a novel approach to screen electron beam-irradiated spice samples; and the authenticity of the results could be confirmed by EPR spectroscopy.
Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2013R1A1A4A03006993).
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independent of radiation processing (Sanyal and Sharma 2009). In the case of all the spice samples, complex EPR spectra were observed, with an increase in the signal intensity of the existing weak singlet (g = 2.006) after the exposure to electron beam at 6, 10, and 14 kGy doses. Raffi and others (1992) and Ahn and others (2013a) previously had reported similar observations, where an intense signal was noticed in the spectrum of irradiated spices; the relatively high intensity was attributed to the irradiation treatment. The line width (Bpp ) of the EPR signal was observed to increase from 0.54 to 0.93 mT possibly because of the induction of multiple paramagnetic centers in the matrix of the samples. The exposure to electron beam leads to another paramagnetic species (triplet signal) at the same g value as that of nonirradiated samples, with a hyperfine coupling constant (hfcc) of 3 mT. This signal was attributed to cellulose radical induced by the ionizing radiation as a consequence of the cleavage of the cellulose polymer chain. The triplet was found to be more prominent in the case of irradiated red chili compared to the cumin samples. Yordanov and Gancheva (2000) and Lee and others (2008) have reported similar observations for γ -irradiated white pepper and sesame seeds, respectively. The signature of the cellulose radical was also found in γ -irradiated soybeans as reported in our earlier communication (Sanyal and Sharma 2009). The triplet signal of the cellulosic radical is a well-established marker for radiation-processed foods according to EN1787 (2000). The effect of increasing the radiation dose on the EPR spectra of the spice samples was studied. At a dose of 14 kGy, the triplet of cellulose was most prominent with maximum signal intensity. The peak to peak intensity of the EPR central line was observed to be significantly related to the radiation dose as shown in Figure 5. The dotted lines represent a linear fit with R2 values of 0.9892, 0.9441, and 0.9702 for EC, RC, and TR samples, respectively. The EPR spectral features of the electron beam-irradiated spice samples were identical to those of the γ -irradiated food samples reported in the literature (Lee and others 2008; Sanyal and Sharma 2009). However, our irradiated spice samples showed distinct changes in EPR spectra with respect to the nonirradiated samples, thus confirming the results obtained with E-nose analysis.
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Sanyal B, Chatterjee S, Variyar PS, Sharma A. 2012. Application of EPR spectroscopy to identify irradiated Indian medicinal plant products. J Food Sci 77:C710–8. Scewartz HM, Bolton JR, Brog DC. 1972. Biological applications of electron spin resonance. New York, NY: Wiley & Sons, Ltd. Tabner BJ, Tabner VA. 1994. Electron spin resonance spectra of γ -irradiated citrus fruits, skin, skin components and stalks. Int J Food Sci Technol 29:143–52. Thayer DW, Josephson ES, Brynjolfsson A, Giddings GG. 1996. Radiation pasteurization of food. Council Agric Sci Technol 7:1–10. Tjaberg TB, Underdal B, Lunde G. 1972. The effect of ionizing radiation on the microbial content and volatile constituents of spices. J Appl Bacteriol 35:473–8.
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von Sonntag C. 1980. Free-radicals reactions of carbohydrates as studied by radiation techniques. Adv Carbohydr Chem Biol 37:7–77. Winquist F, Arwin H, Lund E, Forster R, Day C, Lundstrom I. 1995. Screening of irradiated tomatoes by means of an electronic nose. In Proceedings of Transducers’ 95, Euro sensors IX, the 8th International Conference on Solid- state Sensors and Actuators and Euro sensors IX; June 25–29, pp 691–669; Stockholm, Sweden. Yordanov ND, Gancheva V. 2000. A new approach for extension of the identification period of irradiated cellulose-containing food stuffs by EPR spectroscopy. Appl Radiat Isotopes 52:195–8.
